Epigenetic control of gene expression is a fundamental process required for normal cellular development and maintenance. Abnormal regulation of epigenetic processes, resulting in aberrant histone modification and/or DNA methylation, is a common underlying contributor to many human diseases, including neurological and autoimmune disorders and cancer. The SET-domain protein superfamily of lysine methyltransferases includes critical modulators of gene expression that participate in both histone and DNA methylation. Abnormal SET- protein activity has been shown to contribute to malignant transformation, and recurrent SET-protein mutations have been identified in a wide variety of cancers. Because of the importance of SET-proteins in the regulation of chromatin compaction, DNA methylation, and malignant transformation, our lab has sought to identify and characterize the SET-proteins of the model organism Neurospora crassa. Neurospora has been instrumental as an experimental system for uncovering the connection between histone regulation and DNA methylation, and unique characteristics of Neurospora make it the ideal system for continued discovery of regulators of epigenetic programming. Preliminary studies in our lab indicate that targeted disruption of wild type SET-3 function results in DNA hypermethylation and aberrant methylation spreading. We propose to define SET-3's role in DNA methylation regulation. Our specific aims are: (1) To characterize the genomic interactions of SET- 3; (2) to characterize the lysine methyltransferase activity of SET-3; and (3) to elucidate the function of SET-3 in vivo. Specifically, we will use high-throughput sequencing technology and experimental techniques well established in the Selker lab to map the genomic interaction of SET-3 and characterize the SET-3 regulated transcriptome. We will complement this by mapping DNA methylation across the whole genome in a SET-3- compromised strain. We will then explore the biochemical activity of SET-3 by testing its histone lysine activity in vitro usin a purified catalytic domain construct and by in vivo analyses of DNA and histone methylation patterns. Finally, we will use immunoprecipitation of SET-3 followed by mass spectrometric analysis to identify SET-3-interactors. We will integrate the in vitro activity studies with the in vivo phenotype characterization and protein-protein interaction studies, and place them in the context of SET-3's mapped genomic interactions to derive a global characterization of SET-3 function. These studies should contribute to our understanding of gene expression regulation in eukaryotes, and will aid in our understanding of DNA hypermethylation and methylation spreading in cancer. The long-term goal of our studies is to apply what we have learned to human SET-protein orthologues to inform the development of new targeted cancer therapeutics.